Injury to peripheral nerves can result in significant and permanent functional deficits. Nerve allografts offer a limitless source of nerve graft material that is used clinically to reconstruct severe otherwise irreparable traumatic nerve injuries. FK506 enhances nerve regeneration and is the immunosuppressant of choice for preventing allograft rejection, but can cause significant patient morbidity. The long-term objective of this proposal is to develop strategies to prevent nerve allograft rejection while minimizing the side effects of immunosuppression, thereby improving safety and broadening the clinical indications for peripheral nerve allotransplantation. Donor-specific immune unresponsiveness induced by costimulation blockade has been shown to permit nerve regeneration through allografts in several animal models while maintaining general immunocompetence. Similarly nerve allografts can be cold preserved to decrease antigenicity and seeded with host Schwann cells to facilitate nerve regeneration. A better understanding of the nerve allograft response, the effects of cold preservation, and Schwann cell migration into nerve grafts provides a basis for strategies to minimize requirements for host immunosuppression. Supplementation of nerve allografts with modified host Schwann cells may enhance regeneration through nerve allografts. These approaches will expand the indications for nerve allotransplantation to include less severe injuries where nerve autografts are currently used. In aim 1 a the relative contributions of the direct and indirect pathways of alloantigen presentation in peripheral nerve allografts are characterized using CD4+ and CD8+ knockout and MHC class ll-deficient mice. Aim 1b studies the effects of cold preservation of nerve allografts on these pathways using STAT4 and STAT6 gene knockout mice. In aim 2a, regeneration through nerve allografts is evaluated following simultaneous blockade of CD28/B7 and CD40 costimulatory pathways. In aim 2b, nerve allografts are cold preserved to optimize the efficacy of costimulatory blockade to permit allograft acceptance. In aim 3a, Thy1-CFP/S100-GFP mice are used to characterize Schwann cell migration, differentiation, and maturation when repopulating a cold preserved nerve allograft. In aim 3b, nerve allografts are cold preserved for 7 weeks and then seeded with cultured autologous Schwann cells that overexpress GDNF to permit regeneration through nerve allografts without any immunosuppression. In aim 3c this same construct is evaluated in a long swine allograft model that closely resembles the long nerve defects encountered clinically and will allow translation to the nerve-injured patient.